Lon is an ATP-dependent protease belonging to the “ATPase associated with diverse cellular activities” (AAA+) protein family. In humans, Lon is translated as a precursor and imported into the mitochondria matrix through deletion of the first 114 amino acid residues. In mice, embryonic knockout of lon is lethal. In humans, some dysfunctional lon mutations are tolerated but they cause a developmental disorder known as the CODAS syndrome. To gain a better understanding on the enzymology of human mitochondrial Lon, this study compares the structure-function relationship of the WT versus one of the CODAS mutants R721G to identify the mechanistic features in Lon catalysis that are affected. To this end, steady-state kinetics were used to quantify the difference in ATPase and ATP-dependent peptidase activities between WT and R721G. The K_m values for the intrinsic as well as protein-stimulated ATPase were increased whereas the k_cat value for ATP-dependent peptidase activity was decreased in the R721G mutant. The mutant protease also displayed substrate inhibition kinetics. In vitro studies revealed that R721G did not degrade the endogenous mitochondrial Lon substrate pyruvate dehydrogenase kinase isoform 4 (PDK4) effectively like WT hLon. Furthermore, the pyruvate dehydrogenase complex (PDH) protected PDK4 from hLon degradation. Using hydrogen deuterium exchange/mass spectrometry and negative stain electron microscopy, structural perturbations associated with the R721G mutation were identified. To validate the in vitro findings under a physiologically relevant condition, the intrinsic stability as well as proteolytic activity of WT versus R721G mutant towards PDK 4 were compared in cell lysates prepared from immortalized B lymphocytes expressing the respective protease. The lifetime of PDK4 is longer in the mutant cells, but the lifetime of Lon protein is longer in the WT cells, which corroborate the in vitro structure-functional relationship findings.

Lon 是一种 ATP 依赖性蛋白酶，属于“与多种细胞活动相关的 ATP 酶”(AAA+) 蛋白家族。在人类中，Lon 被翻译为前体，并通过删除前 114 个氨基酸残基导入线粒体基质中。在小鼠中，lon 的胚胎敲除是致命的。在人类中，一些功能失调的 lon 突变是可以耐受的，但它们会导致一种称为 CODAS 综合征的发育障碍。为了更好地了解人类线粒体 Lon 的酶学，本研究比较了 WT 与 CODAS 突变体 R721G 之一的结构-功能关系，以确定受影响的 Lon 催化机制特征。为此，使用稳态动力学来量化 WT 和 R721G 之间 ATP 酶和 ATP 依赖性肽酶活性的差异。在 R721G 突变体中，内在以及蛋白质刺激的 ATP 酶的 K _m值增加，而 ATP 依赖性肽酶活性的 k _cat值降低。突变蛋白酶还表现出底物抑制动力学。体外研究表明，R721G 不像 WT hLon 那样有效降解内源性线粒体 Lon 底物丙酮酸脱氢酶激酶同工型 4 (PDK4)。此外，丙酮酸脱氢酶复合物 (PDH) 保护 PDK4 免遭 hLon 降解。使用氢氘交换/质谱和负染色电子显微镜，鉴定了与 R721G 突变相关的结构扰动。 为了验证生理相关条件下的体外研究结果，在由表达相应蛋白酶的永生化 B 淋巴细胞制备的细胞裂解物中比较了 WT 与 R721G 突变体对 PDK 4 的内在稳定性和蛋白水解活性。 PDK4的寿命在突变细胞中更长，但Lon蛋白的寿命在WT细胞中更长，这证实了体外结构-功能关系的发现。